i) PKC regulates the interaction between nonmuscle myosin and Hgl. The gene product of the Drosophila tumor suppressor gene Discs lethal (2) giant larvae, Dlgl, is a cytoskeletal and plasma membrane-associated protein whose absence leads to neural and imaginal disc tumors. Hgl, the human ortholog of Dlgl, associates with nonmuscle myosin and the interaction is inhibited by a tightly bound kinase. Using a GST-fusion protein which includes a highly conserved sequence containing 4 serine residues, Hgl kinase activity was partially purified from rat brain. Kinase activity was inhibited by the PKC inhibitor GF109203X and PKC was found by immunoblotting to bind tightly to the GST fusion protein. Full-length Hgl expressed in vitro bound to nonmuscle myosin, while a C-terminal fragment truncated distal to the 4 serines did not bind. Phosphorylation of full-length Hgl with PKC and MgATP inhibited binding of Hgl to nonmuscle myosin. We conclude that the interaction between Hgl and nonmuscle myosin can be regulated by PKC-mediated phosphorylation of Hgl, probably at 4 highly conserved serine residues. PKC activation may, thus, have a role in regulating co-localization of Hgl and nonmuscle myosin in response to stimuli. ii) Activation of RBL cells leads to threonine phosphorylation of nonmuscle myosin heavy chain. Antigen activation of IgE-sensitized RBL cells, a rat mast cell line, leads to the rapid threonine phosphorylation of nonmuscle myosin IIA heavy chain, detected with a specific anti-phosphothreonine antibody. Phosphorylation is dependent on extracellular calcium and can be mimicked by addition of the calcium ionophore ionomycin to cells. Cleavage of myosin with hydroxylamine demonstrated that the phosphorylation site is in the C-terminal section of the molecule, while chymotrypsin digestion demonstrated rapid disappearance of labelled phosphothreonine from immunoblots, consistent with a site in the C-terminal non-coiled-coil region. Addition of ionomycin to mouse fibroblasts led to a similar threonine phosphorylation of nonmuscle myosin IIA heavy chain, while no phosphorylation was found in HeLa or Y79 cells, 2 human cell lines. These results are consistent with the site of phosphorylation being Thr 1940, which is conserved in rats and mice, but not in humans; experiments to confirm the threonine localization are underway. - nonmuscle myosin II; protein kinase C; phosphorylation